RyR2 disease mutations at the C-terminal domain intersubunit interface alter closed-state stability and channel activation

Figures

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  Figure 1. C-terminal domain (CTD) intersubunit interface and locations of CTD disease mutations in the 3D structure of RyR2.A, side view of the RyR2 tetramer. B, top view of the RyR2 tetramer. The CTD domain from each subunit is highlighted in red. C and D, the surface presentation of the CTD domain in the closed (C) and open (D) states. Residue P4902 is colored in yellow, E4950 in cyan, and G4955 in green. E and F, transparent CTD surface and ribbon presentation of two neighboring subunits in the closed (E) and open (F) states. Disease-associated CTD mutations are indicated in the same color scheme as in panels C and D. All 3D structure images were generated from Protein Data Bank 5GO9 (closed state) and 5GOA (open state) using PyMOL. G and H, HEK293 cells were transiently transfected with the RyR2 WT or disease-associated CTD mutant complementary DNAs. Immunoblotting of RyR2 WT and mutants from the same amount of transfected cell lysates (G). Note that the RyR2 band with an expected molecular weight of 565 kDa is located inside the separating gel. The expression levels of the WT and disease-associated mutants were normalized to that of GAPDH (one-way ANOVA with Dunnett's post hoc test, F…

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  Figure 2. Effects of disease-associated CTD mutations on [3H]ryanodine binding to RyR2.A and B, [3H]ryanodine binding to cell lysates prepared from HEK293 cells transiently transfected with the RyR2 WT or disease-associated CTD mutant complementary DNAs was carried out at various Ca2+ concentrations (0.1 nM–0.1 mM). The amounts of [3H]ryanodine binding at various Ca2+ concentrations were normalized to the maximal binding (100%). C, EC50 values of Ca2+ activation (one-way ANOVA with Dunnett's post hoc test, F = 50.5, p < 0.0001). D, basal activity (in the near absence of Ca2+, ~0.1 nM) of [3H]ryanodine binding to RyR2 (one-way ANOVA with Dunnett's post hoc test, F = 346.5, p < 0.0001). Data shown are mean ± SD from four to five separate experiments. ∗∗p < 0.01 versus WT. CTD, C-terminal domain; HEK293, human embryonic kidney 293 cells; RyR2, cardiac ryanodine receptor.

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  Figure 3. Effects of disease-associated CTD mutations on caffeine activation of RyR2.A, HEK293 cells were transiently transfected with RyR2 WT or disease-associated CTD mutant complementary DNAs. The fluorescence intensity of the Fluo-3-loaded transfected cells was monitored continuously before and after each caffeine addition. Note that the small drops in fluorescence signal immediately after additions of caffeine are due to dilution of the Fluo-3 fluorescent dye by the added caffeine solution. B, the relationships between caffeine-induced Ca2+ release and cumulative caffeine concentrations in HEK293 cells transfected with RyR2 WT, P4902S, P4902L, E4950K, and G4955E. The amplitude of each caffeine peak was normalized to that of the maximum peak for each experiment. C, the apparent EC50 values of caffeine-induced Ca2+ releases in HEK293 cells transfected with RyR2 WT or mutants (one-way ANOVA with Dunnett's post hoc test, F = 40.7, p < 0.0001). Data shown are mean ± SD from five separate experiments. ∗∗p < 0.01 versus WT. CTD, C-terminal domain; HEK293, human embryonic kidney 293 cells; RyR2, cardiac ryanodine receptor.

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  Figure 4. Effect of disease-associated CTD mutations on SOICR activation and termination thresholds. Stable and inducible HEK293 cell lines expressing RyR2 WT and mutants were transfected with the FRET-based ER luminal Ca2+-sensing protein D1ER 48 h before single-cell FRET imaging. Expression of the RyR2 WT and mutants was induced 24 h before imaging. The cells were perfused with KRH buffer containing increasing levels of extracellular Ca2+ (0–2 mM) to induce SOICR. This was followed by the addition of 2 mM tetracaine to inhibit SOICR and then 20 mM caffeine to deplete the ER Ca2+ stores. Representative FRET recordings from RyR2 WT (A), P4920S (B), P4902L (C), E4950K (D), and G4955E (E) are shown. To minimize the influence of cyan fluorescent protein/yellow fluorescent protein cross talk, relative FRET measurements were used to calculate the activation threshold (F) and termination threshold (G) using the equations shown in A. FSOICR indicates the FRET level at which SOICR occurs, whereas Ftermi represents the FRET level at which SOICR terminates. The fractional Ca2+ release (H) was calculated by subtracting the termination threshold from the activation threshold. The maximum FRET signal…

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  Figure 5. The G4955E mutation markedly increases the channel sensitivity to luminal Ca2+activation. Recombinant RyR2 WT and G4955E mutant channels were partially purified from cell lysate prepared from HEK293 cells transiently transfected with the RyR2 WT or G4955E mutant complementary DNA. Single-channel activities were recorded in a symmetrical recording solution containing 250 mM KCl and 25 mM Hepes (pH 7.4). Representative current traces of single RyR2 WT (A) and G4955E (B) channels in the presence of different cytosolic Ca2+ concentrations and 45 nM luminal Ca2+ (panels a and b) and in the presence of 45 nM cytosolic Ca2+ and different luminal Ca2+ concentrations (panels c and d). The open probability of RyR2 WT or G4955E channels activated by various fixed cytosolic Ca2+ (C) or fixed luminal Ca2+ concentrations (D). The frequency of open events of single RyR2 WT or G4955E mutant channels in the presence of 45 nM cytosolic and 45 nM luminal Ca2+ concentration (E). Data shown are mean ± SD from seven RyR2 WT and seven G4955E single channels. ∗∗p < 0.01 versus WT. HEK293, human embryonic kidney 293 cells; RyR2, cardiac ryanodine receptor.

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  Figure 6. Mutational analyses of the CTD intersubunit interface.A, 3D structure of the U-motif, S6 helix, CTD, and its neighboring U-motif and CTD. B, disease-affected residues (P4902, E4950, and G4955 in green) and negatively and positively charged residues at the CTD intersubunit interface. C, HEK293 cells were transiently transfected with the RyR2 WT or CTD mutant complementary DNAs. Immunoblotting of RyR2 WT and mutants from the same amount of transfected cell lysates. Note that the RyR2 band with an expected molecular weight of 565 kDa is located inside the separating gel. D, the expression levels of the WT and mutants were normalized to that of GAPDH (one-way ANOVA with Dunnett's post hoc test, F = 0.2461, p = 0.9556). Data shown are mean ± SD from four independent experiments. CTD, C-terminal domain; HEK293, human embryonic kidney 293 cells; NS, not significant; RyR2, cardiac ryanodine receptor.

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  Figure 7. Effects of CTD mutations on [3H]ryanodine binding to RyR2. [3H]ryanodine binding to cell lysate prepared from HEK293 cells transiently transfected with the RyR2 WT or CTD mutant complementary DNAs was carried out at various Ca2+ concentrations (0.1 nM–0.1 mM). The amounts of [3H]ryanodine binding at various Ca2+ concentrations were normalized to the maximal binding (100%). [3H]ryanodine binding to RyR2 WT, P4902D, P4902K (A), to RyR2 WT, G4955D, G4955K (B), to RyR2 WT, G4955L (C), or to RyR2 WT and D4956A (D). EC50 values of Ca2+ activation (one-way ANOVA with Dunnett's post hoc test, F = 142.7, p < 0.0001) (E) and basal activity (in the near absence of Ca2+, ~0.1 nM) (F) of [3H]ryanodine binding to RyR2 (one-way ANOVA with Dunnett's post hoc test, F = 71.9, p < 0.0001). Data shown are mean ± SD from four to five separate experiments. ∗∗p < 0.01 versus WT. CTD, C-terminal domain; HEK293, human embryonic kidney 293 cells; RyR2, cardiac ryanodine receptor.

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  Figure 8. Effects of CTD mutations on caffeine activation of RyR2.A, HEK293 cells were transiently transfected with RyR2 WT or mutant complementary DNAs. The fluorescence intensity of the Fluo-3-loaded transfected cells was monitored continuously before and after each caffeine addition. The relationships between caffeine-induced Ca2+ release and cumulative caffeine concentrations in HEK293 cells transfected with RyR2 WT, P4902D, or P4902K (B), with RyR2 WT, G4955K, G4955L, or G4956A (C). D, the apparent EC50 values of caffeine-induced Ca2+ releases in HEK293 cells transfected with RyR2 WT or mutants (one-way ANOVA with Dunnett's post hoc test, F = 149.7, p < 0.0001). Note that the data for RyR2 WT shown here are the same as those shown for Figure 3 as the caffeine response of all mutants shown in these figures was characterized together. Data shown are mean ± SD from four to five separate experiments. ∗∗p < 0.01; ∗p < 0.05 versus WT. CTD, C-terminal domain; HEK293, human embryonic kidney 293 cells; RyR2, cardiac cardiac ryanodine receptor.